Recombinant Actifensin and Defensin-d2 Induce Critical Changes in the Proteomes of Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans

ABSTRACT Drug-resistant strains of Pseudomonas aeruginosa and Candida albicans pose serious threats to human health because of their propensity to cause fatal infections. Defensin and defensin-like antimicrobial peptides (AMPs) are being explored as new lines of antimicrobials, due to their broad range of activity, low toxicity, and low pathogen resistance. Defensin-d2 and actifensin are AMPs from spinach and Actinomyces ruminicola, respectively, whose mechanisms of action are yet to be clearly elucidated. This study investigated the mechanisms of action of the recombinant AMPs through label-free quantitative proteomics. The data are available at PRIDE with accession number PXD034169. A total of 28 and 9 differentially expressed proteins (DEPs) were identified in the treated P. aeruginosa and C. albicans, respectively, with a 2-fold change threshold and P values of <0.05. Functional analysis revealed that the DEPs were involved in DNA replication and repair, translation, and membrane transport in P. aeruginosa, while they were related mainly to oxidative phosphorylation, RNA degradation, and energy metabolism in C. albicans. Protein-protein interactions showed that the DEPs formed linear or interdependent complexes with one another, indicative of functional interaction. Subcellular localization indicated that the majority of DEPs were cytoplasmic proteins in P. aeruginosa, while they were of nuclear or mitochondrial origin in C. albicans. These results show that recombinant defensin-d2 and actifensin can elicit complex multiple organism responses that cause cell death in P. aeruginosa and C. albicans. IMPORTANCE AMPs are considered essential alternatives to conventional antimicrobials because of their broad-spectrum efficacy and low potential for resistance by target cells. In this study, we established that the recombinant AMPs defensin-d2 and actifensin exert proteomic changes in P. aeruginosa and C. albicans within 1 h after treatment. We also found that the DEPs in peptide-treated P. aeruginosa are related to ion transport and homeostasis, molecular functions including nucleic and amino acid metabolism, and structural biogenesis and activity, while the DEPs in treated C. albicans are mainly involved in membrane synthesis and mitochondrial metabolism. Our results also highlight ATP synthase as a potential drug target for multidrug-resistant P. aeruginosa and C. albicans.

between them? 3.P. aeruginosa and C. albicans contains more than 5000 and 6000 proteins, respectively. Why only 276 and 108 proteins was identified in this study, respectively? 4.In table 2, No 1 protein (30S ribosomal protein S6) also up regulated in DPA group, why not included in statistic analysis? 5. In Fig 1, 30S ribosomal protein S4 should be 30S ribosomal protein S6. 6.Line 178-181, based on Fig 1, seven of the downregulated proteins in both treatment showed close relatedness, but not five. 7. In Fig 1 and 2, why the annotation of protein fold change level is different? 8.Line 268, network actifensin-treated (b) should (d). 9.Please rebuild Fig 3 and 4 (c) and (d). It's difficult to understand the result. 10. In Fig 7, please indicate the red and blue meaning. Also, please indicate protein name, but not Protein ID. 11.Line 461, CFU should be given full name when first appears.
Reviewer #2 (Comments for the Author): The manuscript describes the activity of two antimicrobial peptides against multi-drug resistant Pseudomonas aeruginosa and Candida albicans. Considering the great threat that antimicrobial resistance poses and that those two microorganisms are involved in a great number of infections caused by both of them, I think that the manuscript is interesting. However, I missed some extra experiments/data validating the findings from their proteomics analysis. The authors made an extensive analysis of the induced changes in the proteomic landscape in response to both peptides. That leads to several hypotheses regarding their possible mechanisms of action. However, the authors did not try to validate their findings using experimental approaches to investigate the effects of the peptides on both microbes. This would include well-established assays, to evalute membrane or cell wall integrity, electron microscopy to evaluate morphological changes, or assays to evaluate the induction of oxidative stress after the treatments. Omics assays are indeed very good to provide hypotheses, but I really think the authors should try to employ some other experimental approaches to validate their data. In addition to that, the manuscript text is quite extensive in length, and I think it could be shortened to focus on the main changes that were observed. They also need to increase the font size in the figures and I think that a better organization of the figures will improve the manuscript as well.
Staff Comments:

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Response to Reviewer's Comments
Reviewer #1: Comment 1: Line 38 and 39, both peptides bactericidal and fungicidal effects within 1 hour of treatment. I did not find this assay.
Response 1: these results and the methodology for the assay have been published in our earlier study. As such, to prevent dual publication, we have modified by citing the published work. Methodology and results are presented below for review purpose only: Methodology Broth microdilution method was employed to determine the minimum inhibitory concentrations (MIC) of the recombinant peptides against methicillin-resistant S. aureus (MRSA), E. coli, K. pneumoniae, P. aeruginosa and C. albicans. The cell density was adjusted to 0.5 McFarland standard (10 6 CFU/ mL) in normal saline, and C. albicans was further diluted to 10 3 CFU/ mL in Mueller Hinton broth (Oxoid, UK). Then, 10 μL of the inoculum suspension was added into 100 μL of concentrations of recombinant defensin (3.75 -985 µg/mL) or recombinant actifensin (11.5 -2895 µg/mL) diluted in Mueller Hinton broth. Ampicillin was used as positive control for E. coli, K. pneumoniae, P. aeruginosa; vancomycin for MRSA and nystatin for C. albicans. The plates were incubated at 37 °C for 24 h for bacteria or 48 h for C. albicans, then 30 μL of resazurin (0.015%) was added to all wells and further incubated for 2 -4 h to detect microbial activity by colour change from blue to pink. The experiments were performed in triplicates and the minimum inhibitory concentration was determined as the least concentration with no colour change for each organism. To determine the minimum bactericidal/fungicidal concentration (MBC/MFC), a loopful of inoculum from the wells without colour change were plated on Mueller Hinton agar. The plates were then incubated at 37 °C for 24 h. The lowest concentration that showed no colonies was taken as the MBC/MFC. Comment 2: Based on MIC of both peptides, defensin-d2 showed higher activity than actifensin. What's the difference of mode of action between them?
Response 2: from the earlier results of this study, we determined that defensin-d2 showed a broader antimicrobial spectrum compared to actifensin. We postulate that the differences in the activity seen could be attributed to the origin of the antimicrobial peptides and the overall amino acid composition of the peptides. Plant defensins have been studied to exhibit broadspectrum activity against pathogens while bacteriocins exhibit a narrow spectrum activity against bacteria especially Gram positive. Hence, in studies where bacteriocins showed a considerable broader activity, higher concentrations of the bacteriocins were used. While different concentrations are required to elicit cidal actions against P. aeruginosa and C. albicans, similar pattern of interactions are seen for both peptides.
Comment 3: P. aeruginosa and C. albicans contains more than 5000 and 6000 proteins, respectively. Why only 276 and 108 proteins was identified in this study, respectively?
Response 3: the number of rotal proteins identified in this study is attribute to the duration of growth of the test organisms as well as the data acquisition parameters used. Optimal growth is required for optimal protein expression, which in the case of both test organisms will require about 24 hours. But because our focus was in investigating the effect of the peptides at the onset of the log phase, obtaining the entire protein profile of the organisms was not a priority. We believe that the expressed proteins at this stage are very essential for the viability and essential metabolic activities of the organisms. Also, the top 30 parent ions with intensity over 10,000 were set as selection parameters for the dependent data acquisition in order to have representation of the most abundant proteins in the different samples.
The manuscript describes the activity of two antimicrobial peptides against multi-drug resistant Pseudomonas aeruginosa and Candida albicans. Considering the great threat that antimicrobial resistance poses and that those two microorganisms are involved in a great number of infections caused by both of them, I think that the manuscript is interesting.
However, I missed some extra experiments/data validating the findings from their proteomics analysis. The authors made an extensive analysis of the induced changes in the proteomic landscape in response to both peptides. That leads to several hypotheses regarding their possible mechanisms of action. However, the authors did not try to validate their findings using experimental approaches to investigate the effects of the peptides on both microbes. This would include well-established assays, to evaluate membrane or cell wall integrity, electron microscopy to evaluate morphological changes, or assays to evaluate the induction of oxidative stress after the treatments. Omics assays are indeed very good to provide hypotheses, but I really think the authors should try to employ some other experimental approaches to validate their data. In addition to that, the manuscript text is quite extensive in length, and I think it could be shortened to focus on the main changes that were observed. They also need to increase the font size in the figures and I think that a better organization of the figures will improve the manuscript as well.
Response: In an earlier study published, we demonstrated membrane permeabilization of the peptides. We also determined the DNA-binding ability of the peptides by gel retardation assay and UV-Vis. Further we investigated the effects of the peptides on membraneassociated virulence factors. A few of the results are attached here for review purposes only.   We are working on further validating the effects on genomic DNA as well as on transcription of virulence factors as well as genes involved in DNA replication and repair, and as such these data cannot be submitted as part of this manuscript at this time.
As the reviewer rightly mentioned, we agree that omics provides a global insight into possible mechanisms of action of antimicrobials. While our laboratory continues working to experimentally validate these postulates, we believe the content of this manuscript is highly important and sufficient in providing insights into the mechanisms of action of the peptides. We also believe this can generate research interest from other researchers.
Furthermore, the length of the manuscript as well as the figures have been modified to improve the manuscript. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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